Type 2 diabetes currently afflicts >25.8 million Americans and costs the US healthcare system over $245 billion/year. It is a disease characterized by hyperglycemia, hyperinsulinemia, and insulin resistance in skeletal muscle, the primary tissue responsible for insulin-mediated glucose uptake in the body. Despite the importance of muscle in maintaining blood glucose homeostasis, there are currently no pharmaceutical treatments for diabetes that target muscle glucose uptake independent of insulin. Resistance exercise/muscle contractile activity stimulates glucose uptake into muscle; and importantly in type 2 diabetes the ability of exercise to stimulate muscle glucose uptake remains functional. Thus, targeting the mechanisms underlying exercise/contraction-mediated muscle glucose uptake is an effective strategy for lowering blood glucose levels in type 2 diabetes. Unfortunately, these mechanisms are not well understood. The long-term goals of this research are to identify the molecular, cellular or metabolic mechanisms within muscle that regulate insulin-independent glucose uptake and to test whether targeting those mechanisms is effective at ameliorating hyperglycemia in type 2 diabetes. Recent evidence has now implicated the Ca2+-activated, serine/threonine kinase, Ca2+/calmodulin-dependent protein kinase kinase ? (CaMKK?) as a key regulator of exercise-sensitive, insulin-independent muscle glucose uptake, suggesting that CaMKK? may be a promising new target for treating impaired muscle glucose uptake in type 2 diabetes. Unfortunately, the mechanism(s) underlying the ability of CaMKK? to regulate muscle glucose uptake remains unclear. The specific objectives of this proposal are to identify the intracellular signaling protein(s) [i.e. substrate(s)] activated by CaMKK? in muscle; to determine how CaMKK? stimulates muscle glucose utilization; and to determine whether simultaneous activation of an energy consuming process is necessary for sustained CaMKK? -mediated muscle glucose uptake. To achieve these objectives, a combination of state-of-the-art approaches and methodologies will be applied including use of an ATP analog to screen for novel CaMKK? substrates in muscle, in vivo muscle gene transfer/electroporation to allow for the rapid, transient expression of genes in mouse muscle, and generation of a muscle-specific CaMKK? knockout mouse. It is anticipated that the proposed research will elucidate the intracellular mechanism(s) governing the ability of CaMKK? to stimulate glucose uptake in both insulin-sensitive and insulin-resistant muscle; a critical first step towards the development of new treatments for type 2 diabetes aimed at stimulating insulin-independent glucose uptake into skeletal muscle.